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JINGLY FUNG et al.
and 100 mM dCTP with 2.5 III of 1 M TrisHCI, pH 7.5,0.5 III of 0.5 M
EDTA, pH 8.0 and 232 III ultrapure water for a total of250 Ill. Store at
-20
D C. The concentration of nucleoside triphosphates is 2 mM each.
- 1xPBS (Phosphate buffered saline). 0.1 gil CaCh, 0.1 gil MgCh x
6H 2 0,0.2 gil KCl, 0.2 gil KH 2 P0 4 , 8.0 gil NaCl, 2.16 gil Na2HP04 x
7H 2 0.
- 1X PBS/MgCh, add 50 ml of 1 M MgCh to 950 ml 1x PBS.
- SCE: 1 M sorbitol, 0.1 M Na citrate, 10 mM EDTA, pH 7.8.
- SSC: 20xSSC is 3 M NaCI, 0.3 M Na3citrate x 2H 2 0, pH 7.0.
- 4xSSC/0.1% Tween 20: add 100 m120xSSC, 400 ml distilled water and
500 III Tween 20. Mix well.
- lOx Taq buffer: 500 mM KCl, 100 mM TrisHCl, pH 8.3, IS mM MgCh.
Store at -20
D C.
- TBE (Trislborate/EDTA) buffer: lOx TBE is 890 mM Tris base, 890 mM
boric acid, 20 mM EDTA.
- TE (Tris/EDTA) buffer: 1xTE is 10 mM TrisHCI, 1 mM EDTA, pH 7.4,
7.5 or 8.0.
- TE SO buffer: 10 mM TrisHCl, SO mM EDTA, pH 7.8.
- TrisHCI [tris (hydroxymethyl) aminomethane]: 1 M, pH 7.5 or pH 8.0.
Subprotocol 1
Preparation of slides and slide pretreatment
CAUTION: The key elements to consistent hybridization results seem to
depend on (1) conditions during cell preparation and fIxation, (2) relative
humidity, temperature and air flow during preparation of metaphase
spreads, and (3) slide pretreatment with proteolytic agents to enhance
the accessibility of DNA targets to probes without causing undesirable
loss of DNA. We will describe our slide preparation and pretreatment procedures in great detail. The DNA denaturation, hybridization and probe
detection parameters appear to be less important and, consequently, control requirements are critical stringent during those steps.
JINGLY FUNG et al.
and 100 mM dCTP with 2.5 III of 1 M TrisHCI, pH 7.5,0.5 III of 0.5 M
EDTA, pH 8.0 and 232 III ultrapure water for a total of250 Ill. Store at
-20
D C. The concentration of nucleoside triphosphates is 2 mM each.
- 1xPBS (Phosphate buffered saline). 0.1 gil CaCh, 0.1 gil MgCh x
6H 2 0,0.2 gil KCl, 0.2 gil KH 2 P0 4 , 8.0 gil NaCl, 2.16 gil Na2HP04 x
7H 2 0.
- 1X PBS/MgCh, add 50 ml of 1 M MgCh to 950 ml 1x PBS.
- SCE: 1 M sorbitol, 0.1 M Na citrate, 10 mM EDTA, pH 7.8.
- SSC: 20xSSC is 3 M NaCI, 0.3 M Na3citrate x 2H 2 0, pH 7.0.
- 4xSSC/0.1% Tween 20: add 100 m120xSSC, 400 ml distilled water and
500 III Tween 20. Mix well.
- lOx Taq buffer: 500 mM KCl, 100 mM TrisHCl, pH 8.3, IS mM MgCh.
Store at -20
D C.
- TBE (Trislborate/EDTA) buffer: lOx TBE is 890 mM Tris base, 890 mM
boric acid, 20 mM EDTA.
- TE (Tris/EDTA) buffer: 1xTE is 10 mM TrisHCI, 1 mM EDTA, pH 7.4,
7.5 or 8.0.
- TE SO buffer: 10 mM TrisHCl, SO mM EDTA, pH 7.8.
- TrisHCI [tris (hydroxymethyl) aminomethane]: 1 M, pH 7.5 or pH 8.0.
Subprotocol 1
Preparation of slides and slide pretreatment
CAUTION: The key elements to consistent hybridization results seem to
depend on (1) conditions during cell preparation and fIxation, (2) relative
humidity, temperature and air flow during preparation of metaphase
spreads, and (3) slide pretreatment with proteolytic agents to enhance
the accessibility of DNA targets to probes without causing undesirable
loss of DNA. We will describe our slide preparation and pretreatment procedures in great detail. The DNA denaturation, hybridization and probe
detection parameters appear to be less important and, consequently, control requirements are critical stringent during those steps.